Substrates for Improved Live-Cell Fluorescence Labeling of SNAP-tag
Substrates for Improved Live-Cell Fluorescence Labeling of SNAP-tag
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DOI：
10.2174/1381612811319300011
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发表时间：
2013-09-01
影响因子：
3.1
通讯作者：
Xu, Ming-Qun
中科院分区：
文献类型：
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作者：
Correa, Ivan R., Jr.;Baker, Brenda;Xu, Ming-Qun
SNAP标签标记技术为融合蛋白成像提供了一种简单、稳健和通用的方法，可用于广泛的实验应用。由于标记反应的特异性和共价性，SNAP标签非常适合使用荧光显微镜技术分析和定量融合靶蛋白。在这份报告中，我们提出了我们最近的研究结果，在体外和细胞培养中的SNAP标签融合蛋白的标签与SNAP标签基板来自单一区域异构体的羧基罗丹明染料。羧基罗丹明是生物技术应用中非常宝贵的荧光染料，包括DNA测序、微阵列检测和荧光原位杂交。我们发现SNAP-标签优先与羧基罗丹明荧光染料的6-位置区域异构体反应，而5-区域异构体主要贡献于背景荧光。我们的实验研究还表明，苄基氯嘧啶（CP）共轭6-羧基罗丹明表现出显着增加的信号-噪声比的荧光标记的细胞蛋白质相比，苄基鸟嘌呤（BG）共轭物，大概是由于更高的细胞渗透性。这些基于纯6-区域异构体的新SNAP标签底物可以显着提高活细胞中的荧光标记，并应成为生物成像应用的有力工具。
The SNAP-tag labeling technology provides a simple, robust, and versatile approach to the imaging of fusion proteins for a wide range of experimental applications. Owing to the specific and covalent nature of the labeling reaction, SNAP-tag is well suited for the analysis and quantification of fused target protein using fluorescence microscopy techniques. In this report, we present our most recent findings on the labeling of SNAP-tag fusion proteins both in vitro and in cell culture with SNAP-tag substrates derived from single regioisomers of carboxyrhodamine dyes. Carboxyrhodamines are invaluable fluorescent dyes for biotechnology applications including DNA sequencing, detection on microarrays, and fluorescence in situ hybridization. We found that SNAP-tag reacts preferentially with the 6-positional regioisomer of carboxyrhodamine fluorescent dyes, whereas the 5-regioisomer predominantly contributes to background fluorescence. Our experimental study also indicates that benzylchloropyrimidine (CP) conjugates of 6-carboxyrhodamines exhibit a dramatic increase in the signal-to-noise ratio of fluorescently labeled cellular proteins compared to the benzylguanine (BG) conjugates, presumably due to higher cell permeability. These new SNAP-tag substrates based on pure 6-regioisomers can significantly improve fluorescence labeling in live cells and should become powerful tools for bioimaging applications.